Many extracellular and secreted proteins are post-translationally modified with glycans, including N-acetylgalactosamine (GalNAc)-type O-linked glycans. This common form of protein glycosylation is initiated by addition of GalNAc to oxygen atoms of serine or threonine residues. GalNAc-type O-linked glycosylation plays functional roles in diverse biological processes including mucin assembly, developmental signaling, human genetic disorders, cell-cell adhesion events, and cancer progression. Despite playing essential roles, mechanistic understanding of the functions of GalNAc-type O-linked glycosylation is incomplete, due in part to the inadequacy of tools available to probe and control this modification. To meet this challenge, we will carry out a high-throughput screening (HTS) campaign to discover small molecules that inhibit the polypeptide GalNAc-transferase (ppGalNAcT) family of enzymes, responsible for adding GalNAc residues to proteins to initiate GalNAc-type O-linked glycan biosynthesis. Our primary HTS assay is a mass spectrometry-based assay using purified ppGalNAcT1 enzyme and a mucin- derived peptide as a glycosylation substrate. We will screen the 330,000-compound collection from the UT Southwestern HTS core facility, including commercial compounds and natural product fractions. Compounds that show potent and dose-dependent inhibition of ppGalNAcTs in vitro will be evaluated for ppGalNAcT inhibition in cells. We will also use a plate-based assay to test whether hit molecules inhibit mucin secretion from respiratory epithelial cells, a process that depends on GalNAc-type O-linked glycans. Candidate inhibitors that display cell-based activity will be further analyzed by a cascade of assays aimed at testing the mechanism of action and selectivity of inhibition. Structure-activity relationship (SAR) analysis will be performed on top compounds. A handful of high-performing inhibitors will be subjected to a full molecular characterization including analysis of kinetic mechanism, profiling of induced transcriptional and glycosylation changes, measuring affinity of ppGalNAcT binding, structural characterization of the inhibitor-ppGalNAcT complex, and a proof-of-concept experiment testing effects on the migratory behavior of lung cancer cells. At the end of the granting period, we aim to identify one or more pharmacophores that provide potent and selective inhibition of the ppGalNAcT family in vitro and in cells. In addition to their utility as chemical probes for academic research, these compounds may have the potential to serve as lead molecules for new therapeutic approaches to treat cancers, such as pancreatic cancer and non-small cell lung cancer (NSCLC), and respiratory disease, including asthma, that are characterized by mucus overproduction.